Synovial calprotectin: A potentially useful biomarker for the diagnosis of Kingella kingae native arthritis

Abstract Kingella kingae is a bacteria involved in developing arthritis in children. Its diagnosis remains difficult. We report a case for which a new biomarker, calprotectin measured in the synovial fluid, was strongly positive and a specific molecular test was the only way to diagnose it specifically.


| INTRODUCTION
Acute osteoarticular infections in children represent medical and surgical emergencies with potentially severe functional sequelae. 1 Hence, these infections require a rapid and sensitive diagnosis. Molecular diagnostic tools have dramatically improved the diagnostic yield of pediatric septic arthritis (SA). However, more than 20% of these infections remain unconfirmed because no microorganism is identified. 2 In such situations, the use of inflammatory markers may be helpful to quickly confirm the diagnosis. Serum C-reactive protein (CRP) level could be a useful tool, even if it is commonly lower during Kingella kingae infectionsthe leading causative agent in young children's arthritis. 2 Here we report a case of K. kingae knee arthritis with a high level of synovial calprotectin, an inflammatory biomarker recently successfully assessed in prosthetic joint infection (PJI). 3,4

| CASE REPORT
An otherwise healthy 14-month-old child was admitted to the pediatric emergency department after a 2-day history of fever and right-knee pain leading to lameness. Physical examination revealed an irritable joint with a limited range of motion and swelling. Ultrasound pointed out a joint effusion. Laboratory results showed an increase in serum CRP value (38.6 mg/L, reference range 0-5 mg/L), whereas a complete blood cell count was within normal limits. Knee SA was therefore suspected.
A joint aspiration and an intensive washing with a saline solution were performed. Amoxicillin and clavulanic acid (150 mg per kg of body weight per day) was injected intravenously immediately after surgery. Joint aspiration yielded 5 mL of a purulent fluid sent for cytological and microbiological analysis. A pediatric blood culture vial (Peds Plus/F Becton Dickinson) was promptly inoculated with synovial fluid (SF) in the operating room. White blood cell count showed 142,080 cells/mm 3 with 91% neutrophils. Unfortunately, Gram staining did not reveal any microorganisms. The joint fluid was inoculated on blood and Polyvitex chocolate agar plates (bioMérieux) incubated for 7 days at 37°C in 5% CO 2 . SF CRP level was measured at 11.7 mg/L. Interestingly, we had an opportunity to assess the SF calprotectin level (a biomarker released from neutrophils upon their activation) which was increased, higher than 300 mg/L (Lysftone), as shown in Figure 1. At that stage, as preliminary laboratory results were in accordance with the SA suspicion, antibiotics were continued.
Despite an optimized culture protocol, every microbiological culture remained sterile after 7 days of incubation. DNA extraction and 16S rRNA gene amplification with universal primers 27f and 1492r were performed directly from the fluid, as previously described. 5 Despite a 16S rRNA amplification, 16S rRNA gene sequencing failed. Given the age of the patient, a new PCR assay selectively targeting the nonribosomal gene cpn60 from K. kingae was performed and came back positive, confirming the diagnosis. 6 The clinical outcome was promptly favorable, and the serum CRP level decreased within normal limits 3 days after surgery. Considering recommendations for bone and joint infections in children of her age, 1 the antibiotic was switched to oral route (amoxicillin/clavulanic acid 500 mg three times per day) and the child was discharged to home on postoperative Day 3. Antibiotic therapy was discontinued 4 weeks after the surgery. At that time, clinical examination and plain radiographs were strictly normal. A favorable outcome without sequelae or any relapse was observed after a 3-month follow-up period.

| DISCUSSION
Kingella kingae, a Gram-negative bacterium belonging to the HACEK group, is the leading cause of pediatric osteoarticular infections (mostly SA) in children younger than 4 years. 1,2 The available published experience on K. kingae arthritis pointed out that isolation of this bacterium is challenging. The use of an automated blood culture system enhances the recovery of K. kingae from joint fluids. 7 Most recent studies have demonstrated the obvious superiority of molecular methods, in particular PCR targeting K. kingae-specific genes (cpn60 or rtxA genes), that of K. kingae arthritis diagnosis. 6 In total, these results convincingly demonstrate that (i) the isolation of K. kingae remains complicated, even when specimens are seeded into blood culture vials; and that (ii) molecular diagnostic tools increase the detection rate. However, despite the use of molecular methods, more than 20% of pediatric osteoarticular infections persist unconfirmed because no microorganism is detected. 2 Therefore, other simple tools are needed for acute painful joint diagnosis. Among serum inflammatory markers, procalcitonin shows better diagnosis performances than serum CRP and appears to be the most accurate biomarker in SA, both in adult and pediatric populations. However, due to a serious lack of sensitivity, procalcitonin is not suitable for excluding the diagnosis of SA. 8 Synovial CRP has been suggested as a useful parameter for the diagnosis of PJI in adults with a relevant cutoff around 10 mg/L, and has even been included in international scoring systems. 9 Unfortunately, no data are available regarding SF CRP among pediatric SA. In our case, SF CRP was barely above the cutoff.
On the contrary, synovial calprotectin, an important proinflammatory factor of innate immunity released from activated granulocytes, was very high. Recently, synovial calprotectin potential value in the diagnosis of PJI has been stressed. 3,4 A cutoff of 50 mg/L has been suggested as adequate to diagnose a PJI. Indeed, this fast (15 min), quantitative, and easy to do and read test (by using a smartphone application) can be performed simply in a routine lab. Regarding native joints, few studies recently highlighted that synovial calprotectin could be a relevant biomarker to discriminate SA from other inflammatory arthritis. This new biomarker may help clinicians to decide whether antibiotic therapy is needed or not in conjunction with clinical, radiological, and microbiological findings. 10 To conclude, we report a case of native SA due to K. kingae in a 14-month-old child. Despite an optimized culture protocol including a blood culture vial, the negative culture could have led to a diagnostic wandering. The availability (in just a few minutes) of a synovial calprotectin new test highlights its potential usefulness to distinguish juvenile idiopathic arthritis from native joint arthritis. Therefore, this biomarker should be assessed prospectively in this context.